Humanized antibodies that recognize alpha-synuclein

ABSTRACT

The present application discloses humanized 9E4 antibodies. The antibodies bind to human alpha synuclein and can be used for immunotherapy of Lewy body disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 15/857,104, now U.S.Pat. No. 10,450,369 filed Dec. 28, 2017, which is a divisional of U.S.Ser. No. 15/387,580 filed Dec. 21, 2016 now U.S. Pat. No. 9,884,906,which is a divisional of U.S. Ser. No. 14/156,441 filed Jan. 15, 2014now U.S. Pat. No. 9,556,259, which is a continuation of PCT/US12/62290,filed Oct. 26, 2012, which claims priority to U.S. 61/553,131 filed Oct.28, 2011, and U.S. 61/711,208, filed Oct. 8, 2012, each of which isincorporated by reference in the entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

The sequence listing written in file 531251SEQLIST.txt is 35,340 bytesand was created on May 16, 2019. The information contained in this fileis hereby incorporated by reference.

BACKGROUND

Synucleinopathies also known as Lewy body diseases (LBDs), arecharacterized by degeneration of the dopaminergic system, motoralterations, cognitive impairment, and formation of Lewy bodies (LBs)and/or Lewy neurites. (McKeith et al., Neurology (1996) 47:1113-24).Synucleinopathies include Parkinson's disease (including idiopathicParkinson's disease), Diffuse Lewy Body Disease (DLBD) also known asDementia with Lewy Bodies (DLB), Lewy body variant of Alzheimer'sdisease (LBV), Combined Alzheimer's and Parkinson disease, pureautonomic failure and multiple system atrophy (MSA; e.g.,Olivopontocerebellar Atrophy, Striatonigral Degeneration and Shy-DragerSyndrome). Several nonmotor signs and symptoms are thought to beharbingers for synucleinopathies in the prodromal phase of the diseases(i.e, the presymptomatic, subclinical, preclinical, or premotor period).Such early signs include, for example, REM sleep behavior disorder(RBD), loss of smell and constipation (Mahowald et al., Neurology (2010)75:488-489). Lewy body diseases continue to be a common cause formovement disorders and cognitive deterioration in the aging population(Galasko et al., Arch. Neurol. (1994) 51:888-95).

Alpha-synuclein is part of a large family of proteins including beta-and gamma-synuclein and synoretin. Alpha-synuclein is expressed in thenormal state associated with synapses and is believed to play a role inneural plasticity, learning and memory. Several studies have implicatedalpha-synuclein with a central role in PD pathogenesis. The protein canaggregate to form insoluble fibrils in pathological conditions. Forexample, synuclein accumulates in LBs (Spillantini et al., Nature (1997)388:839-40; Takeda et al., J. Pathol. (1998) 152:367-72; Wakabayashi etal., Neurosci. Lett. (1997) 239:45-8). Mutations in the alpha-synucleingene co-segregate with rare familial forms of parkinsonism (Kruger etal., Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science (1997)276:2045-7). Over expression of alpha synuclein in transgenic mice(Masliah et al., Science (2000) 287:1265-9) and Drosophila (Feany etal., Nature (2000) 404:394-8) mimics several pathological aspects ofLewy body disease. In addition, it has been suggested that solubleoligomers of synuclein may be neurotoxic (Conway K A, et al., Proc NatlAcad Sci USA (2000) 97:571-576; Vol1esMJ, Lansbury P T, Jr Biochemistry(2003) 42:7871-7878). The accumulation of alpha-synuclein with similarmorphological and neurological alterations in species and animal modelsas diverse as humans, mice, and flies suggests that this moleculecontributes to the development of Lewy body disease.

SUMMARY OF THE CLAIMED INVENTION

The invention provides antibodies comprising a mature humanized heavychain variable region comprising the three Kabat CDRs of SEQ ID NO:11,and being at least 90% identical to SEQ ID NO:11, and a humanized lightchain comprising the three Kabat CDRs of SEQ ID NO:4, and being at least90% identical to SEQ ID NO:4. In some antibodies, the mature heavy chainvariable region is at least 95%, 96%, 97%, 98%, or 99% identical to SEQID NO:11 and mature light chain variable region is at least 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO:4. In some antibodies positionL36 (Kabat numbering) can be occupied by Y or F, position L83 (Kabatnumbering) can be occupied by F or L, position H73 (Kabat numbering) canbe occupied by N or D and position H93 (Kabat numbering) can be occupiedby A or S. In some of such antibodies the amino acid sequence of themature heavy chain variable region is otherwise that of SEQ ID NO:11 andthe amino acid sequence of the mature light chain variable region isotherwise that of SEQ ID NO:4.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence designated SEQ ID NO:8 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:3. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:8 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:4. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:8 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:5. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:9 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:3. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:9 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:4. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:9 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:5. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:10 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:3. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:10 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:4. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:10 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:5. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:11 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:3. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:11 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:4. In someantibodies, the mature heavy chain variable region has an amino acidsequence designated SEQ ID NO:11 and the mature light chain variableregion has an amino acid sequence designated SEQ ID NO:5.

The invention further provides an antibody comprising a humanized heavychain comprising the three Kabat CDRs of SEQ ID NO:11 and a humanizedlight chain comprising the three CDRs of SEQ ID NO:4 provided thatposition L36 (Kabat numbering) is occupied by F or Y and/or position L83(Kabat numbering) is occupied by L or F and/or position H73 (Kabatnumbering) is occupied by D or N, and/or position H93 (Kabat numbering)is occupied by S or A. In some such antibodies, position L36 (Kabatnumbering) is occupied by F and position H73 (Kabat numbering) isoccupied by D, and position H93 (Kabat numbering) is occupied by S. Insome such antibodies, position L36 is occupied by F. In some suchantibodies, position L83 is occupied by L. In some such antibodiesposition H73 is occupied by D. In some such antibodies, position H93 isoccupied by A. In some such antibodies, position L36 is occupied by Fand position L83 is occupied by L. In some such antibodies, position L36is occupied by F and position H73 is occupied by D. In some suchantibodies, position L36 is occupied by F and position H93 is occupiedby A. In some such antibodies, position L36 is occupied by F, positionL83 is occupied by L and position H73 is occupied by D. In some suchantibodies, position L36 is occupied by F, position L83 is occupied by Land position H93 is occupied by A. In some such antibodies, position L36 is occupied by F, position L83 is occupied by L, position 1173 isoccupied by D and position H93 is occupied by A. In some suchantibodies, residues at positions L36, L83, H73 and H93 (Kabatnumbering) are occupied by amino acids as indicated in Table 1 isoccupied by F and position H73 (Kabat numbering) is occupied by D, andposition H93 (Kabat numbering) is occupied by A. In some suchantibodies, position L36 (Kabat numbering) is occupied by F and positionH93 (Kabat numbering) is occupied by S. In some such antibodies,position H73 (Kabat numbering) is occupied by D and position H93 (Kabatnumbering) is occupied by S. In some such antibodies, position 1173(Kabat numbering) is occupied by D and position 1193 (Kabat numbering)is occupied by A. In some such antibodies, position H93 (Kabatnumbering) is occupied by S. In some such antibodies, position 1173(Kabat numbering) is occupied by N. In some such antibodies, positionL36 (Kabat numbering) is occupied by F, position L83 (Kabat numbering)is occupied by L, position H73 (Kabat numbering) is occupied by D, andposition H93 (Kabat numbering) is occupied by S. In some suchantibodies, position L36 (Kabat numbering) is occupied by F, positionL83 (Kabat numbering) is occupied by L and position H93 (Kabatnumbering) is occupied by S.

In any of the above antibodies, the mature heavy chain variable regioncan be fused to a heavy chain constant region and the mature light chainconstant region can be fused to a light chain constant region.

In any of the above antibodies, the heavy chain constant region can be amutant form of natural human constant region which has reduced bindingto an Fcγ receptor relative to the natural human constant region.

In any of the above antibodies, the heavy chain constant region can beof human IgG1 isotype. In some antibodies the allotype is G1m3. In someantibodies, the allotype is G1m1.

The invention further provides a nucleic acid encoding any of theabove-mentioned mature heavy chain variable regions and/or any of theabove-mentioned mature light chain variable region, e.g., SEQ ID NO:15,17, 18, 19, and 20.

The invention further provides a host cell comprising a vectorcomprising any of the nucleic acids described above.

The invention further provides a method of treating a patient having orat risk of a Lewy body disease, comprising administering to the patientan effective regime of any of the above-mentioned antibodies. In somemethods, the disease is Parkinson's disease. In some methods, decline ofcognitive function in the patient is inhibited. In some methods,neuritic and/or axonal alpha synuclein aggregates are reduced. In somemethods, neuritic dystrophy in the patient is reduced. In some methods,synaptic and/or dendritic density is preserved. In some methods, themethod preserves synaptophysin and/or MAP2 in the patient.

The invention further provides a method of treating a patient having orat risk of synucleinopathy, comprising administering to the patient aneffective regime of any of the above-mentioned antibodies. In somemethods, the disease is Parkinson's disease. In some methods, thedisease is REM sleep behavior disorder (RBD). In some methods, thedisease is Dementia with Lewy Bodies (DLB) or multiple system atrophy(MSA). In some methods, decline of cognitive function in the patient isinhibited. In some methods, neuritic and/or axonal alpha synucleinaggregates are reduced. In some methods, neuritic dystrophy in thepatient is reduced. In some methods, synaptic and/or dendritic densityis preserved. In some methods, the method preserves synaptophysin and/orMAP2 in the patient.

The invention further provides methods of detecting Lewy bodies in apatient having or at risk of a Lewy body disease, comprisingadministering to the patient an effective amount of any of theabove-mentioned antibodies, wherein the antibody binds to Lewy bodiesand bound antibody is detected. In some methods, the disease isParkinson's disease. In some methods, the disease is Dementia with LewyBodies (DLB) or multiple system atrophy (MSA). In some methods, theantibody is labeled.

The invention further provides a method of reducing Lewy body formationin a patient having or at risk of a Lewy body disease, comprisingadministering to the patient an effective amount of any of theabove-mentioned antibodies. In some methods, the disease is Parkinson'sdisease. In some methods, the disease is Dementia with Lewy Bodies (DLB)or multiple system atrophy (MSA). In some methods, decline of cognitivefunction in the patient is inhibited. In some methods, neuritic and/oraxonal alpha synuclein aggregates are reduced. In some methods, neuriticdystrophy in the patient is reduced. In some methods, synaptic and/ordendritic density is preserved. In some methods, the method preservessynaptophysin and/or MAP2 in the patient.

The invention further provides a method of inhibiting synucleinaggregation or clearing Lewy bodies or synuclein aggregates in a patienthaving or at risk of a Lewy body disease, comprising administering tothe patient an effective amount of any of the above-mentionedantibodies. In some methods, the disease is Parkinson's disease. In somemethods, the disease is Dementia with Lewy Bodies (DLB) or multiplesystem atrophy (MSA). In some methods, decline of cognitive function inthe patient is inhibited. In some methods, neuritic and/or axonal alphasynuclein aggregates are reduced. In some methods, neuritic dystrophy inthe patient is reduced. In some methods, synaptic and/or dendriticdensity is preserved. In some methods, the method preservessynaptophysin and/or MAP2 in the patient.

The invention further provides a pharmaceutical composition comprisingany of the above-mentioned antibodies.

The invention further provides a method of producing an antibody,comprising culturing cells transformed with nucleic acids encoding theheavy and light chains of the antibody, so that the cell secrete theantibody; and purifying the antibody from cell culture media; whereinthe antibody is any of the antibodies described above.

The invention further provides a method producing a cell line producingan antibody, comprising introducing a vector encoding heavy and lightchains of an antibody and a selectable marker into cells; propagatingthe cells under conditions to select for cells having increased copynumber of the vector; isolating single cells from the selected cell; andbanking cells cloned from a single cell selected based on yield ofantibody; wherein the antibody is any of the antibodies described above.Some such methods further comprises propagating the cells underselective conditions and screening for cell lines naturally expressingand secreting at least 100 mg/L/10⁶ cells/24 h.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the amino acid sequences of the parentalmurine mAb (referred to as m9E4) with the humanized 9E4 heavy chainmature variable region. 1791009Hu9E4VHFr (SEQ ID NO:7) is human acceptorV_(H) sequence. CDR regions according to Kabat definition are underlinedand in bold.

FIG. 2 shows an alignment of the amino acid sequences of the parentalmurine mAb (referred to as m9E4) with the humanized 9E4 light chainmature variable region. 63102889Hu9E4VLFr (SEQ ID NO:2) is humanacceptor V_(L) sequence. CDR regions according to Kabat definition areunderlined and in bold.

FIG. 3 shows the results of passive immunotherapy with 9E4 on memoryperformance in probe portion of the Morris water maze test.

FIG. 4 shows the results of passive immunotherapy with 9E4 on speed anderrors in the round beam test.

FIG. 5 shows immunoprecipitation of various versions of humanized 9E4antibodies towards its antigen taken from diseased tissue. Ch9E4:chimeric 9E4; H1L3: Hu9E4VHv1-Hu9E4VLv3; H3L2: Hu9E4VHv3-Hu9E4VLv2; H3L3: Hu9E4VHv3-Hu9E4VLv3; N.S.=non-specific.

FIG. 6 shows Western blotting of recombinant human synuclein with mouse,chimeric and humanized 9E4 antibodies.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of m9E4VL variable region.

SEQ ID NO:2 is the amino acid sequence of 63102889Hu9E4VLFr variableregion.

SEQ ID NO:3 is the amino acid sequence of Hu9E4VLv1 variable region.

SEQ ID NO:4 is the amino acid sequence of Hu9E4VLv2 variable region.

SEQ ID NO:5 is the amino acid sequence of Hu9E4VLv3 variable region.

SEQ ID NO:6 is the amino acid sequence of m9E4VH variable region.

SEQ ID NO:7 is the amino acid sequence of 1791009Hu9E4VHFr variableregion.

SEQ ID NO:8 is the amino acid sequence of Hu9E4VHv1 variable region.

SEQ ID NO:9 is the amino acid sequence of Hu9E4VHv2 variable region.

SEQ ID NO:10 is the amino acid sequence of Hu9E4VHv3 variable region.

SEQ ID NO:11 is the amino acid sequence of Hu9E4VHv4 variable region.

SEQ ID NO:12 is the amino acid sequence of natural human wild-typealpha-synuclein.

SEQ ID NO:13 is the amino acid sequence of humanized 9E4 light chainconstant region, with Arginine at the N-terminus.

SEQ ID NO:14 is the amino acid sequence of humanized 9E4 heavy chainconstant region.

SEQ ID NO:15 is the nucleotide sequence of Hu9E4VLv1 variable region.

SEQ ID NO:16 is the nucleotide sequence of Hu9E4VLv2 variable region.

SEQ ID NO:17 is the nucleotide sequence of Hu9E4VLv3 variable region.

SEQ ID NO:18 is the nucleotide sequence of Hu9E4VHv1 variable region.

SEQ ID NO:19 is the nucleotide sequence of Hu9E4VHv2 variable region.

SEQ ID NO:20 is the nucleotide sequence of Hu9E4VHv3 variable region.

SEQ ID NO:21 is the nucleotide sequence of Hu9E4VHv4 variable region.

SEQ ID NO:22 is the amino acid sequence of Hu9E4VL signal peptide.

SEQ ID NO:23 is the nucleotide sequence of Hu9E4VL signal peptide.

SEQ ID NO:24 is the amino acid sequence of Hu9E4VH signal peptide.

SEQ ID NO:25 is the nucleotide sequence of Hu9E4VH signal peptide.

SEQ ID NO:26 is the Hu9E4VL consensus amino acid sequence.

SEQ ID NO:27 is the Hu9E4VH consensus amino acid sequence.

SEQ ID NO:28 is the amino acid sequence of humanized 9E4 light chainconstant region, without the Arginine at the N-terminus.

SEQ ID NO:29 is the amino acid sequence of the version 3humanized 9E4light chain comprising (a) a variable region and (b) a constant regionwith Arginine at the N-terminus.

SEQ ID NO:30 is the amino acid sequence of the version 3humanized 9E4light chain comprising (a) a variable region and (b) a constant regionwithout the Arginine at the N-terminus.

SEQ ID NO:31 is the amino acid sequence of the version 3 humanized 9E4heavy chain comprising a variable region and a constant region.

SEQ ID NO:32 is the amino acid sequence of the BIP version of humanized9E4 heavy chain Glm3 allotype constant region.

Definitions

Monoclonal antibodies are typically provided in isolated form. Thismeans that an antibody is typically at least 50% w/w pure of proteinsand other macromolecules arising from its production or purification butdoes not exclude the possibility that the monoclonal antibody iscombined with an excess of pharmaceutical acceptable carrier(s) or othervehicle intended to facilitate its use. Sometimes monoclonal antibodiesare at least 60%, 70%, 80%, 90%, 95 or 99% w/w pure of proteins andother macromolecules from production or purification.

Specific binding of a monoclonal antibody to its target antigen means anaffinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific bindingis detectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces. Specific bindingdoes not however necessarily imply that a monoclonal antibody binds oneand only one target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes variable regionof about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7, incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabatalso provides a widely used numbering convention (Kabat numbering) inwhich corresponding residues between different heavy chains or betweendifferent light chains are assigned the same number (e.g., H83 meansposition 83 by Kabat numbering in the mature heavy chain variableregion; likewise position L36 means position 36 by Kabat numbering inthe mature light chain variable region).

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target includingseparate heavy chains, light chains Fab, Fab′, F(ab′)₂, F(ab)c,diabodies, Dabs, nanobodies, and Fv. Fragments can be produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes a bispecificantibody and/or a humanized antibody. A bispecific or bifunctionalantibody is an artificial hybrid antibody having two differentheavy/light chain pairs and two different binding sites (see, e.g.,Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990);Kostelny et al., J. Immunol. 148:1547-53 (1992)). In some bispecificantibodies, the two different heavy/light chain pairs include ahumanized 9E4 heavy chain/light chain pair and a heavy chain/light chainpair specific for a different epitope on alpha synuclein than that boundby 9E4. Humanized antibodies are discussed generally below in Section IVB.

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined by X-ray crystallography ofthe antibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% but preferably 75%,90% or 99% as measured in a competitive binding assay. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.

A “patient” includes a human or other mammalian subject that receiveseither prophylactic or therapeutic treatment.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises antibody may contain the antibody alone or incombination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within the SEM of a stated value.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “symptom” refers to a subjective evidence of a disease, such asaltered gait, as perceived by the patient. A “sign” refers to objectiveevidence of a disease as observed by a physician.

Statistical significance means p≤0.05.

“Cognitive function” refers to mental processes such as any or all ofattention, memory, producing and understanding language, solvingproblems, and making an interest in one's surroundings and self-care.

“Enhanced cognitive function” or “improved cognitive function” refers toimprovement relative to a baseline, for example, diagnosis or initiationof treatment. “Decline of cognitive function” refers to a decrease infunction relative to such a base line.

In animal model systems such as rat or mouse, cognitive function may bemeasured by methods using a maze in which subjects use spatialinformation (e.g, Morris water maze, Barnes circular maze, elevatedradial arm maze, T maze and others), fear conditioning, activeavoidance, illuminated open-field, dark activity meter, elevatedplus-maze, two-compartment exploratory test or forced swimming test.

In humans, cognitive function can be measured by one or more of severalstandardized tests. Examples of a test or assay for cognitive functionwere described (Ruoppila, l. and Suutama, T. Scand. J. Soc. Med. Suppl.53, 44-65, 1997) and include standardized psychometric tests (e. g.Wechsler Memory Scale, the Wechsler Adult Intelligence Scale, Raven'sStandard Progressive Matrices, Schaie-Thurstone Adult Mental AbilitiesTest), neuropsychological tests (e. g. Luria-Nebraska), metacognitiveself-evaluations (e. g. Metamemory Questionnaire), visual-spatialscreening tests (e. g. Poppelreuter's Figures, Clock Recognition,Honeycomb Drawing and Cancellation), cognitive screening tests (e. g.Folstein's Mini Mental State Test) and reaction time tests. Otherstandard tests for cognitive performance include the Alzheimer's DiseaseAssessment Scale-cognitive subscale (ADAS-cog); the clinical globalimpression of change scale (CIBIC-plus scale); the Alzheimer's DiseaseCooperative Study Activities of Daily Living Scale (ADCS-ADL); the MiniMental State Exam (MMSE); the Neuropsychiatric Inventory (NPI); theClinical Dementia Rating Scale (CDR); the Cambridge NeuropsychologicalTest Automated Battery (CANTAB) or the Sandoz ClinicalAssessment-Geriatric (SCAG), Stroop Test, Trail Making, Wechsler DigitSpan, and the CogState computerized cognitive test. In addition,cognitive function may be measured using imaging techniques such asPositron Emission Tomography (PET), functional magnetic resonanceimaging (fMRJ), Single Photon Emission Computed Tomography (SPECT), orany other imaging technique that allows one to measure brain function.

DETAILED DESCRIPTION

I. General

The invention provides humanized 9E4 antibodies. The antibodies areuseful for treatment and diagnoses of a Lewy body disease.

II. Target Molecules

Natural human wildtype alpha-synuclein is a peptide of 140 amino acidshaving the following amino acid sequence:

(SEQ ID NO: 12) MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYVGSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQKTVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

(Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90:11282-6).; GenBankaccession number: P37840). The protein has three recognized domains, aKTKE repeat domain covering amino acids 1-61, a NAC (Non-amyloidcomponent) domain running from about amino acids 60-95, and a C-terminalacidic domain running from about amino acid 98 to 140.

Unless otherwise apparent from the context, reference to alpha-synucleinor its fragments includes the natural human wildtype amino acidsequences indicated above, and human allelic variants thereof,particularly those associated with Lewy body disease (e.g., E46K, A30Pand A53T, with the first letter indicates the amino acid in SEQ IDNO:12, the number is the codon position in SEQ ID NO:12, and the secondletter is the amino acid in the allelic variant). Such variants canoptionally be present individually or in any combination in any of theaspect of the invention described below. The induced mutations E83Q,A90V, A76T, which enhance alpha synuclein aggregation, can also bepresent individually or in combination with each other and/or humanallelic variants E46K, A30P and A53T.

III. Lewy Body Diseases

Lewy Body Diseases (LBD) are characterized by degeneration of thedopaminergic system, motor alterations, cognitive impairment, andformation of Lewy bodies (LBs). (McKeith et al., Neurology (1996)47:1113-24). Lewy Bodies are spherical protein deposits found in nervecells. Their presence in the brain disrupts the brain's normal functioninterrupting the action of chemical messengers including acetylcholineand dopamine. Lewy Body diseases include Parkinson's disease (includingidiopathic Parkinson's disease), Diffuse Lewy Body Disease (DLBD) alsoknown as Dementia with Lewy Bodies (DLB), Lewy body variant ofAlzheimer's disease (LBV), Combined Alzheimer's and Parkinson diseaseand as multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy,Striatonigral Degeneration and Shy-Drager Syndrome). DLBD sharessymptoms of both Alzheimer's and Parkinson's disease. DLBD differs fromParkinson's disease mainly in the location of Lewy Bodies. In DLBD LewyBodies form mainly in the cortex. In Parkinson's disease, they formmainly in the substantia nigra. Other Lewy Body diseases include PureAutonomic Failure, Lewy body dysphagia, Incidental LBD, and InheritedLBD (e.g., mutations of the alpha-synuclein gene, PARK3 and PARK4).

IV. Antibodies of the Invention

A. Binding Specificity and Functional Properties

Humanized antibodies of the invention specifically bind to human alphasynuclein. The affinity of some humanized antibodies (i.e., Ka) ispreferably within a factor of five or two of that of the mouse antibody9E4. Some humanized antibodies have an affinity that is the same (withinexpermental error) or greater than that of the mouse 9E4 antibody.Preferred humanized antibodies bind to the same epitope and/or competewith the mouse antibody 9E4 for binding to human alpha synuclein.

In some antibodies, humanized 9E4 forms one arm of a bispecificantibody, the other arm of which is an antibody that binds to a receptorexpressed on the blood brain barrier, such as an insulin receptor, aninsulin-like growth factor (IGF) receptor, a leptin receptor, or alipoprotein receptor, or preferably a transferrin receptor (Friden etal., PNAS 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993).Such a bispecific antibody can be transferred cross the blood brainbarrier by receptor-mediated transcytosis. Brain uptake of thebispecific antibody can be further enhanced by engineering thebi-specific antibody to reduce its affinity to the blood brain barrierreceptor. Reduced affinity for the receptor resulted in a broaderdistributioin in the brain (see, e.g., Atwal. et al. Sci. Trans. Med. 3,84ra43, 2011; Yu et al. Sci. Trans. Med. 3, 84ra44, 2011).

Exemplary bispecific antibodies can also be (1) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg(2010)); (2) a Tandab, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (3) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(4) a so called “dock and lock” molecule, based on the “dimerization anddocking domain” in Protein Kinase A, which, when applied to Fabs, canyield a trivalent bispecific binding protein consisting of two identicalFab fragments linked to a different Fab fragment; (5) a so-calledScorpion molecule, comprising, e.g., two scFvs fused to both termini ofa human Fc-region. Examples of platforms useful for preparing bispecificantibodies include but are not limited to BiTE (Micromet), DART(MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor) orDuoBody (based on Fab arm exchange, Genmab).

B. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen et al., U.S. Pat. Nos. 5,530,101and 5,585,089; Winter et al., U.S. Pat. No. 5,225,539, Carter, U.S. Pat.No. 6,407,213, Adair, U.S. Pat. No. 5,859,205 6,881,557, Foote, U.S.Pat. No. 6,881,557). The acceptor antibody sequences can be, forexample, a mature human antibody variable region sequence, a compositeof such sequences, a consensus sequence of human antibody sequences(e.g., light and heavy chain variable region consensus sequences ofKabat, 1991, supra), or a germline variable region sequence. A preferredacceptor sequence for the heavy chain is the human mature heavy chainvariable region with NCBI accession code AAC50998 (GI: 1791009) or othermature heavy chain variable region derived from germline IGHV3-7′01 orIGHV3-7′02 (clones name V3-7 or VH3-11) (Glas et al., Clin Exp Immunol.107:372-80, 1997) or a mature heavy chain variable region sequenceincorporating one of these germ line sequences. For the light chain, apreferred acceptor sequence is the light chain mature variable regionwith NCBI accession code AAY33350 (GI:63102889) or other mature lightchain sequence derived from the germline IGKV1D-39 or IGKV1-39 (clonename 02 or 012) (Kramer et al., Eur J Immunol. 35:2131-45, 2005) or alight chain mature variable region sequence incorporating one of thesegerm line sequences. Thus, a humanized antibody of the invention is anantibody having three light chain and three heavy chain CDRs as definedby Kabat from the donor 9E4 antibody and mature variable regionframework sequences and constant regions, if present, entirely orsubstantially from human antibody sequences. Likewise a humanized heavychain is a heavy chain having three heavy chain CDRs as defined by Kabatfrom the heavy chain of the 9E4 antibody, and a mature heavy chainvariable sequence and heavy chain constant region sequence, if present,entirely or substantially from human antibody heavy chain sequence.Likewise a humanized light chain is a light chain having three lightchain CDRs as defined by Kabat from the light chain of the 9E4 antibody,and a mature light chain variable sequence and light chain constantregion sequence, if present, entirely or substantially from humanantibody light chain sequence. The mature variable region frameworksequences of an antibody chain or the constant region sequence of anantibody chain are substantially from a human mature variable regionframework sequence or human constant region sequence respectively whenat least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of correspondingresidues defined by Kabat are identical.

Certain amino acids from the human mature variable region frameworkresidues can be selected for substitution based on their possibleinfluence on CDR conformation and/or binding to antigen. Investigationof such possible influences is by modeling, examination of thecharacteristics of the amino acids at particular locations, or empiricalobservation of the effects of substitution or mutagenesis of particularamino acids.

For example, when an amino acid differs between a murine mature variableregion framework residue and a selected human mature variable regionframework residue, the human framework amino acid can be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g. is within about        6 Å of a CDR region)    -   (4) mediates interaction between the heavy and light chains.

The invention provides humanized forms of the mouse 9E4 antibodyincluding three exemplified humanized light chain mature variableregions (Hu9E4VLv1-v3; SEQ ID NOs:3-5) and four exemplified humanizedheavy chain mature variable regions (Hu9E4VHv1-v4; SEQ ID NOs:8-11). SEQID NO:4 includes the three Kabat CDRs of the mouse 9E4 light chain andthe mature variable region frameworks of AAY33350. SEQ ID NOS. 3 and 5include backmutations as shown in Table 2. SEQ ID NO. 11 includes thethree Kabat CDRs of mouse 9E4 and the mature variable region frameworksof AAC50998. SEQ ID NOs:8-10 include backmutations as shown in Table 3.

The invention provides variants of the humanized 9E4 antibody in whichthe humanized heavy chain mature variable region shows at least 90%, 95%or 99% identity to SEQ ID NOs:8-11 and the humanized light chain maturevariable region shows at least 90, 95 or 99% sequence identity to SEQ IDNOs:3-5, but in which any variation from the designated SEQ ID NO.occurs in a mature variable region framework rather than a Kabat CDR. Insome such antibodies, position L36 is occupied by Y or F, and/orposition L83 is occupied by F or L, and/or position H73 is occupied by Nor D and/or position H93 is occupied by A or S (all positions here, aselsewhere, in this application are by Kabat numbering). In some suchantibodies, some or all of the backmutations in Hu9E4VLv1-v3 andHu9E4VHv1-v4 are retained. In other words, one or both of heavy chainpositions H73 and H93 is occupied by D and A respectively. Likewise insome antibodies one or both of light chain positions L36 and L83 isoccupied by F and L respectively. In some antibodies, 1, 2, 3 or allfour of positions H73, H93, L36 and L83 is/are occupied by D, A, F and Lrespectively. In some antibodies, 0, 1, or 2 positions are changed inthe heavy chain mature variable region framework relative to SEQ IDNO:11, and 0, 1, or 2 positions are change in the light chain maturevariable region framework relative to SEQ ID NO:4.

Some antibodies comprise a humanized heavy chain comprising the threeKabat CDRs of SEQ ID NO:11 and a humanized light chain comprising thethree Kabat CDRs of SEQ ID NO:4 provided that position L36 (Kabatnumbering) is occupied by F or Y and/or position L83 (Kabat numbering)is occupied by L or F and/or position H73 (Kabat numbering) is occupiedby D or N, and/or position H93 (Kabat numbering) is occupied by S or A.In some such antibodies, position L36 (Kabat numbering) is occupied byF. In some such antibodies, position L36 (Kabat numbering) is occupiedby F and position L83 (Kabat numbering) is occupied by L. In some suchantibodies, position L36 (Kabat numbering) is occupied by F and positionH73 (Kabat numbering) is occupied by D. In some such antibodies,position L36 (Kabat numbering) is occupied by F and position H93 (Kabatnumbering) is occupied by S. In some such antibodies, position L36(Kabat numbering) is occupied by F and position H93 (Kabat numbering) isoccupied by A. In some such antibodies, position L36 (Kabat numbering)is occupied by F, position L83 (Kabat numbering) is occupied by L, andposition H73 (Kabat numbering) is occupied by D. In some suchantibodies, position L36 (Kabat numbering) is occupied by F, positionL83 (Kabat numbering) is occupied by L, and position H93 (Kabatnumbering) is occupied by S. In some such antibodies, position L36(Kabat numbering) is occupied by F, position L83 (Kabat numbering) isoccupied by L, and position H93 (Kabat numbering) is occupied by A. Insome such antibodies, position L36 (Kabat numbering) is occupied by F,position H73 (Kabat numbering) is occupied by D, and position H93 (Kabatnumbering) is occupied by S. In some such antibodies, position L36(Kabat numbering) is occupied by F, position L83 is occupied by F,position H73 (Kabat numbering) is occupied by D, and position H93 (Kabatnumbering) is occupied by S. In some such antibodies, position L36(Kabat numbering) is occupied by F, position H73 (Kabat numbering) isoccupied by D, and position H93 (Kabat numbering) is occupied by A. Insome such antibodies, position L36 (Kabat numbering) is occupied by F,position L83 (Kabat numbering) is occupied by L, position H73 (Kabatnumbering) is occupied by D, and position H93 (Kabat numbering) isoccupied by S. In some such antibodies, position L36 (Kabat numbering)is occupied by F, position L83 (Kabat numbering) is occupied by L,position H73 (Kabat numbering) is occupied by D, and position H93 (Kabatnumbering) is occupied by A. In some such antibodies, position L83(Kabat numbering) is occupied by L. In some such antibodies, positionL83 (Kabat numbering) is occupied by L and position H73 (Kabatnumbering) is occupied by D. In some such antibodies, position L83(Kabat numbering) is occupied by L and position H93 (Kabat numbering) isoccupied by S. In some such antibodies, position L83 (Kabat numbering)is occupied by L and position H93 (Kabat numbering) is occupied by A. Insome such antibodies, position L83 (Kabat numbering) is occupied by L,position H73 (Kabat numbering) is occupied by D, and position H93 (Kabatnumbering) is occupied by S. In some such antibodies, position L83(Kabat numbering) is occupied by L, position H73 (Kabat numbering) isoccupied by D, and position H93 (Kabat numbering) is occupied by A. Insome such antibodies, position H73 (Kabat numbering) is occupied by D.In some such antibodies, position H73 (Kabat numbering) is occupied by Dand position H93 (Kabat numbering) is occupied by S. In some suchantibodies, position H73 (Kabat numbering) is occupied by D and positionH93 (Kabat numbering) is occupied by A. In some such antibodies,position H93 (Kabat numbering) is occupied by S. In some suchantibodies, position H93 (Kabat numbering) is occupied by A. In somesuch antibodies, position L36 is occupied by Y, position L83 is occupiedby F, position H73 is occupied by N and position H93 is occupied by S.Some exemplary antibodies with desirable residues at positions L36, L83,H73, and H93 and combinations thereof are listed in Table 1 below:

TABLE 1 Exemplary antibodies with desirable residues at positions L36,L83, H73, and H93 (Kabat numbering). Exemplary Antibody L36 L83 H73 H931 F F N A 2 F L N A 3 F F D A 4 F F N S 5 (version 3) F L D A 6 F L N S7 (version 1) F F D S 8 F L D S 9 Y L N A 10 Y L D A 11 Y L N S 12 Y L DS 13 Y F D A 14 Y F D S 15 (version 2)  Y F N S

In some antibodies, the heavy chain mature variable region has an aminoacid sequence designated SEQ ID NO:10. In some antibodies, the lightchain mature variable region has an amino acid sequence designated SEQID NO:5 or SEQ ID NO:3. In some such antibodies, the heavy chain maturevariable region has an amino acid sequence designated SEQ ID NO:10, andthe light chain mature variable region has an amino acid sequencedesignated SEQ ID NO:5 or SEQ ID NO:3. In some such antibodies, theheavy chain mature variable region has an amino acid sequence designatedSEQ ID NO:10, and the light chain mature variable region has an aminoacid sequence designated SEQ ID NO:5.

Other amino acid substitutions can be made in the mature variable regionframework, for example, in residues not in contact with the CDRs. Oftenthe replacements made in the variant humanized sequences areconservative with respect to the replaced amino acids. In someantibodies, replacements relative to Hu9E4VLv1-v3 and Hu9E4VHv1-v4(whether or not conservative) have no substantial effect on the bindingaffinity or potency of the resultant antibody relative to Hu9E4VLv1-v3and Hu9E4VHv1-v4, that is, its ability to bind human alpha synuclein.

Variants typically differ from the heavy and light chain mature variableregion sequences of Hu9E4VLv1-v3 and Hu9E4VHv1-v4 by a small number(e.g., typically no more than 1, 2, 3, 5 or 10 in either the light chainor heavy chain mature variable region framework, or both) ofreplacements, deletions or insertions.

C. Selection of Constant Region

The heavy and light chain variable regions of humanized antibodies canbe linked to at least a portion of a human constant region. The choiceof constant region depends, in part, whether antibody-dependentcell-mediated cytotoxicity, antibody dependent cellular phagocytosisand/or complement dependent cytotoxicity are desired. For example, humanisotopes IgG1 and IgG3 have complement-dependent cytotoxicity and humanisotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce strongercell mediated effector functions than human IgG2 and IgG4. Light chainconstant regions can be lambda or kappa. An exemplary human light chainkappa constant region has the amino acid sequence of SEQ ID NO:13. Somesuch light chain kappa constant regions can be encoded by a nucleic acidsequence. The N-terminal arginine of SEQ ID NO:13 can be omitted, inwhich case light chain kappa constant region has the amino acid sequenceof SEQ ID NO:28. Some such light chain kappa constant regions can beencoded by a nucleic acid sequence. An exemplary human IgG1 heavy chainconstant region has the amino acid sequence of SEQ ID NO:14 (with orwithout the C-terminal lysine). Some such heavy chain constant regionscan be encoded by a nucleic acid sequence. Antibodies can be expressedas tetramers containing two light and two heavy chains, as separateheavy chains, light chains, as Fab, Fab′, F(ab′)2, and Fv, or as singlechain antibodies in which heavy and light chain mature variable domainsare linked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype bind to a non-polymorphic region of a one or more otherisotypes. Thus, for example, another heavy chain constant region is ofIgG1 Glm3 allotype and has the amino acid sequence of SEQ ID NO:32. Yetanother heavy chain constant region has the amino acid sequence of SEQID NO:32 except that it lacks the C-terminal lysine.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering is used in this paragraphfor the constant region) for increasing the half-life of an antibody.Substitution at any or all of positions 234, 235, 236 and/or 237 reduceaffinity for Fcγ receptors, particularly FcγRI receptor (see, e.g., U.S.Pat. No. 6,624,821). Some antibodies have alanine substitution atpositions 234, 235 and 237 of human IgG1 for reducing effectorfunctions. Optionally, positions 234, 236 and/or 237 in human IgG2 aresubstituted with alanine and position 235 with glutamine (see, e.g.,U.S. Pat. No. 5,624,821).

D. Expression of Recombinant Antibodies

Antibodies can be produced by recombinant expression. Nucleic acidsencoding the antibodies can be codon-optimized for expression in thedesired cell-type (e.g., CHO or Sp2/0). Recombinant nucleic acidconstructs typically include an expression control sequence operablylinked to the coding sequences of antibody chains, includingnaturally-associated or heterologous promoter regions. The expressioncontrol sequences can be eukaryotic promoter systems in vectors capableof transforming or transfecting eukaryotic host cells. Once the vectorhas been incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies. The vector or vectors encoding the antibody chains can alsocontain a selectable gene, such as dihydrofolate reductase, to allowamplification of copy number of the nucleic acids encoding the antibodychains.

E. coli is a prokaryotic host particularly useful for expressingantibodies, particularly antibody fragments. Microbes, such as yeast arealso useful for expression. Saccharomyces is a preferred yeast host,with suitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilizations.

Mammalian cells can be used for expressing nucleotide segments encodingimmunoglobulins or fragments thereof. See Winnacker, From Genes toClones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines, various COS celllines, HeLa cells, HEK293 cells, L cells, and non-antibody-producingmyelomas including Sp2/0 and NS0. It can be advantageous to use nonhumancells. Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, an enhancer(Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Suitable expression control sequences are promoters derived fromendogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected of FACS-based single-cell cloning to generatemonoclonal lines. Specific productivities above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, can be advantageous. Antibodies produced by single cell clonescan also be tested for turbidity, filtration properties, PAGE, IEF, UVscan, HP-SEC, carboydrate-oligosaccharide mapping, mass spectrometery,and bining assay, such as ELISA or Biacore. A selected clone can then bebanked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, columnchromatography (e.g., hydrophobic interaction or ion exchange), low-pHfor viral inactivation and the like (see generally, Scopes, ProteinPurification (Springer-Verlag, N.Y., 1982)).

Methodology for commercial production of antibodies including codonoptimization, selection of promoters, transcription elements, andterminators, serum-free single cell cloning, cell banking, use ofselection markers for amplification of copy number, CHO terminator,serum free single cell cloning, improvement of protein titers (see,e.g., U.S. Pat. Nos. 5,786,464, 6,114,148, 6,063,598, 7,569,339,WO2004/050884, WO2008/012142, WO2008/012142, WO2005/019442,WO2008/107388, and WO2009/027471, and U.S. Pat. No. 5,888,809).

V. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above. Typically, the nucleic acids alsoencode a signal peptide fused to the mature heavy and light chains(e.g., signal peptides having amino acid sequences of SEQ ID NOS: 22 and24 that can be encoded by SEQ ID NOS: 23 and 25). Coding sequences onnucleic acids can be in operable linkage with regulatory sequences toensure expression of the coding sequences, such as a promoter, enhancer,ribosome binding site, transcription termination signal and the like.The nucleic acids encoding heavy and light chains can occur in isolatedform or can be cloned into one or more vectors. The nucleic acids can besynthesized by for example, solid state synthesis or PCR of overlappingoligonucleotides. Nucleic acids encoding heavy and light chains can bejoined as one contiguous nucleic acid, e.g., within an expressionvector, or can be separate, e.g., each cloned into its own expressionvector.

VI. Therapeutic Applications

The invention provides several methods of treating or effectingprophylaxis of Lewy Body disease in patients suffering from or at riskof such disease. Patients amenable to treatment include individuals atrisk of disease of a LBD but not showing symptoms, as well as patientspresently showing symptoms or the early warning signs ofsynucleinopathies, for example, EEG slowing, neuropsychiatricmanifestations (depression, dementia, hallucinations, anxiety, apathy,anhedonia), autonomic changes (orthostatic hypotension, bladderdisturbances, constipation, fecal incontinence, sialorrhea, dysphagia,sexual dysfunction, changes in cerebral blood flow), sensory changes(olfactory, pain, color discrimination abnormal sensations), sleepdisorders (REM sleep behavior disorder (RBD), restless legssyndrome/periodic extremity movements, hypersomnia, insomnia) andmiscellaneous other signs and symptoms (fatigue, diplopia, blurredvision, seborrhea, weight loss/gain). Therefore, the present methods canbe administered prophylactically to individuals who have a known geneticrisk of a LBD. Such individuals include those having relatives who haveexperienced this disease, and those whose risk is determined by analysisof genetic or biochemical markers. Genetic markers of risk toward PDinclude mutations in the alpha-synuclein or Parkin, UCHLI, and CYP2D6genes; particularly mutations at positions 30 and 53 of thealpha-synuclein gene. Individuals presently suffering from Parkinson'sdisease can be recognized from its clinical manifestations includingresting tremor, muscular rigidity, bradykinesia and posturalinstability.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to a therapeutic agent(e.g., a truncated form of alpha-synuclein peptide) over time. If theresponse falls, a booster dosage is indicated.

Antibodies can be used for treating or effecting prophylaxis of LewyBody disease in patients by administration under conditions thatgenerate a beneficial therapeutic response in a patient (e.g., reductionof neuritic and/or axonal alpha synuclein aggregates, reduction ofneuritic dystrophy, improving cognitive function, and/or reversing,treating or preventing cognitive decline) in the patient. In somemethods, the areas of neuritic dystrophy in the neuropil of neocortexand/or basal ganglia can be reduced by on average at least 10%, 20%,30%, or 40% in treated patients compared with a control population.

Cognitive impairment, progressive decline in cognitive function, changesin brain morphology, and changes in cerebrovascular function arecommonly observed in patients suffering from or at risk of Lewy Bodydisease. Administration of the present antibodies can inhibit or delaydecline of cognitive function in such patients.

The invention also provides methods of preserving or increasing synapticdensity and/or dentritic density. An index of changes in synaptic ordentritic density can be measured by markers of synapse formation(synaptophysin) and/or dendrites (MAP2). In some methods, the synapticor dentritic density can be restored to the level of synaptic ordentritic density in a healthy subject. In some methods, the mean levelof synaptic or dentritic density in treated patients can be elevated by5%, 10%, 15%, 20%, 25%, 30% or more as compared to a population ofuntreated control patients.

VII. Pharmaceutical Compositions and Methods of Treatment

In prophylactic applications, an antibody or agent for inducing anantibody or a pharmaceutical composition the same is administered to apatient susceptible to, or otherwise at risk of a disease in a regime(dose, frequency and route of administration) effective to reduce therisk, lessen the severity, or delay the onset of at least one sign orsymptom of the disease. In some prophylactic applications, the regime iseffective to inhibit or delay accumulation of alpha synuclein andtruncated fragments in the brain, and/or inhibit or delay its toxiceffects and/or inhibit/or delay development of behavioral deficits. Intherapeutic applications, an antibody or agent to induce an antibody isadministered to a patient suspected of, or already suffering from a Lewybody disease in a regime (dose, frequency and route of administration)effective to ameliorate or at least inhibit further deterioration of atleast one sign or symptom of the disease. In some therapeuticapplications, the regime is effective to reduce or at least inhibitfurther increase of levels of alpha synuclein and truncated fragments,associated toxicities and/or behavioral deficits.

A regime is considered therapeutically or prophylactically effective ifan individual treated patient achieves an outcome more favorable thanthe mean outcome in a control population of comparable patients nottreated by methods of the invention, or if a more favorable outcome isdemonstrated in treated patients versus control patients in a controlledclinical trial (e.g., a phase II, phase II/III or phase III trial) atthe p<0.05 or 0.01 or even 0.001 level.

Effective doses vary depending upon many different factors, includingmeans of administration, target site, physiological state of the patientincluding type of Lewy body disease, whether the patient is an ApoEcarrier, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.

An exemplary dosage range for antibodies is from about 0.01 to 5 mg/kg,and more usually 0.1 to 3 mg/kg or 0.15-2 mg/kg or 0.15-1.5 mg/kg, ofpatient body weight. Antibody can be administered such doses daily, onalternative days, weekly, fortnightly, monthly, quarterly, or accordingto any other schedule determined by empirical analysis. An exemplarytreatment entails administration in multiple dosages over a prolongedperiod, for example, of at least six months. Additional exemplarytreatment regimes entail administration once per every two weeks or oncea month or once every 3 to 6 months.

Antibodies can be administered via a peripheral route (i.e., one inwhich an administered or induced antibody crosses the blood brainbarrier to reach an intended site in the brain. Routes of administrationinclude topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intrathecal, intraperitoneal, intranasal or intramuscular.Some routes for administration of antibodies are intravenous andsubcutaneous. This type of injection is most typically performed in thearm or leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection.

Pharmaceutical compositions for parenteral administration are can besterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The present regimes can be administered in combination with anotheragent effective in treatment or prophylaxis of the disease beingtreated. For example, in the case of Parkinson's disease, immunotherapyagainst alpha synuclein WO/2008/103472, Levodopa, dopamine agonists,COMT inhibitors, MAO-B inhibitors, Amantadine, or anticholinergic agentscan be used in combination with the present regimes.

VIII. Other Applications

The antibodies described above can be used for detecting alpha-synucleinin the context of clinical diagnosis or treatment or in research. Theantibodies can also be sold as research reagents for laboratory researchin detecting cells bearing alpha-synuclein and their response to variousstimuli. In such uses, monoclonal antibodies can be labeled withfluorescent molecules, spin-labeled molecules, enzymes or radioisotypes,and can be provided in the form of kit with all the necessary reagentsto perform the assay for alpha-synuclein. The antibodies can also beused to purify alpha-synuclein, e.g., by affinity chromatography.

The antibodies can be used for detecting LBs in a patient. Such methodsare useful to diagnose or confirm diagnosis of PD, or other diseaseassociated with the presence of LBs in the brain, or susceptibilitythereto. For example, the methods can be used on a patient presentingwith symptoms of dementia. If the patient has LBs, then the patient islikely suffering from a Lewy body disease, such as Parkinson's disease.The methods can also be used on asymptomatic patients. Presence of Lewybodies or other abnormal deposits of alpha-synuclein indicatessusceptibility to future symptomatic disease. The methods are alsouseful for monitoring disease progression and/or response to treatmentin patients who have been previously diagnosed with a Lewy body disease.

The methods can be performed by administering an antibody and thendetecting the antibody after it has bound. If desired, the clearingresponse can be avoided by using an antibody fragment lacking afull-length constant region, such as a Fab. In some methods, the sameantibody can serve as both a treatment and diagnostic reagent.

For diagnosis (e.g., in vivo imaging), the antibodies can beadministered by intravenous injection into the body of the patient, ordirectly into the brain by intracranial injection or by drilling a holethrough the skull. The dosage of reagent should be within the sameranges as for treatment methods. Typically, the antibody is labeled,although in some methods, the antibody is unlabelled and a secondarylabeling agent is used to bind to the antibody. The choice of labeldepends on the means of detection. For example, a fluorescent label issuitable for optical detection. Use of paramagnetic labels is suitablefor tomographic detection without surgical intervention. Radioactivelabels can also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size and/or intensity oflabeled loci to corresponding base line values. The base line values canrepresent the mean levels in a population of undiseased individuals.Base line values can also represent previous levels determined in thesame patient. For example, base line values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the base line values. A decrease in values relative tobase line signals a positive response to treatment.

The antibodies can be used to generate anti-idiotype antibodies. (see,e.g., Greenspan & Bona, FASEB J. 7(5):437-444, 1989; and Nissinoff, J.Immunol. 147:2429-2438, 1991). Such anti-idiotype antibodies can beutilized in pharmacokinetics, pharmacodynamics, biodistribution studiesas well as in studies of clinical human-anti-human antibody (HAHA)responses in individuals treated with the antibodies. For example,anti-idiotypic antibodies bind specifically the variable region ofhumanized 9E4 antibodies and therefore can be used to detect humanized9E4 antibodies in pharmacokinetic studies and help to quantifyhuman-anti-human antibody (HAHA) responses in treated individuals.

All patent filings, website, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example I. Design of Humanized 9E4 Antibodies

The starting point or donor antibody for humanization is the mouseantibody 9E4 produced by the hybridoma having ATCC Accession No.PTA-8221 and described in U.S. patent application Ser. No. 11/710,248(publication number US2009/0208487). The variable kappa (Vκ) of 9E4belongs to mouse Kabat subgroup 1 which corresponds to human Kabatsubgroup 1. The variable heavy (Vh) of 9E4 belongs to mouse Kabatsubgroup 3d which corresponds to human Kabat subgroup 3 (Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition. NIHPublication No. 91-3242, 1991). Kabat numbering is used throughout inthis Example.

The 17-residue CDR-L1 belongs to canonical class 3, the 7-residue CDR-L2belongs to class 1, and the 9-residue CDR-L3 belongs to class 1 in Vk(Martin & Thornton, J Mol Biol. 263:800-15, 1996). The 5-residue CDR-H1belongs to class 1, and the 17-residue CDR-H2 belongs to class 2 (Martin& Thornton, J Mol Biol. 263:800-15, 1996). CDR-H3 has no canonicalclasses, but the 7 residue loop probably has a kinked base according tothe rules of Shirai et al. (FEBS Lett. 455:188-97, 1999).

A search was made over the protein sequences in the PDB database(Deshpande et al., Nucleic Acids Res. 33: D233-7, 2005) to findstructures which would provide a rough structural model of 9E4. Thecrystal structure of dimeric antibody X836 (pdb code 3MBX) (Teplyakov etal, Mol Immunol., 47(14):2422-6, 2010) was chosen for the Vκ structuresince it had good resolution (1.6 A) and overall sequence similarity to9E4 Vκ, retaining the same canonical structures for the loops. 1H3P(Pizarro et al., FEBS Lett. 509:463-8, 2001) was used for the Vhstructure. It had good overall sequence similarity and reasonableresolution (2.6 A), but also had the same length CDR-H3 with a kinkedbase. In addition, CDRs-H1 and H2 had the same canonical structures as9E4 Vh. DeepView/Swiss-PdvViewer 3.7 (SP5) (Guex and Peitsch,Electrophoresis 18: 2714-2723, 1997) was used structure modeling.

A search of the non-redundant protein sequence database from NCBIallowed selection of suitable human frameworks into which to graft themurine CDRs. For Vκ, a human kappa light chain with NCBI accession codeAAY33350 (GI:63102889) (Kramer et al., Eur J Immunol. 35:2131-45, 2005)was chosen. It has the same canonical classes for CDR-L2 and L3 as 9E4,and belongs to human germline IGKV1D-39 or IGKV1-39 (clone name 02 or012) according to IMGT convention. It is a member of Kabat human kappasubgroup 1. For Vh, human Ig heavy chain with NCBI accession codeAAC50998 (GI:1791009) (Glas et al., Clin Exp Immunol. 107:372-80, 1997)was chosen, again with the same canonical classes as 9E4 and belongingto human germline IGHV3-7′01 or IGHV3-7′02 (clones name V3-7 or VH3-11).It is a member of Kabat human heavy subgroup 3.

The following positions differing between the human acceptor and mousedonor variable region frameworks were identified as being candidates forbackmutation. H73 is on the edge of the antigen binding site andinteracts with CDR-H2. H93 is an interface residue that lies beneath theCDR-H1 and H3 loops. L36 is a Vκ/Vh interface residue. L83 is in closeproximity to the constant domain. In 9E4, L83 is a leucine, whereas inhuman framework, L83 is a larger amino acid phenylalanine.

Three humanized heavy chains and three humanized light chains are madeincorporating back mutations at different permutations of thesepositions (FIG. 1A, B, sequence alignment, and Tables 2-3).

TABLE 2 V_(H) Backmutations V_(H) variant V_(H) exon acceptor sequencedonor framework residues Hu9E4VHv1 NCBI accession code H73, H93 AAC50998Hu9E4VHv2 NCBI accession code H93 AAC50998 Hu9E4VHv3 NCBI accession codeH73 AAC50998

TABLE 3 V_(L) Backmutations V_(L) variant V_(L) exon acceptor sequencedonor framework residues Hu9E4VLv1 NCBI accession code L36 AAY33350Hu9E4VLv2 NCBI accession code None AAY33350 Hu9E4VLv3 NCBI accessioncode L36, L83 AAY33350

>9E4Vκ Version1 (SEQ ID NO: 3) DIQMTQSPSSLSASVGDRVTITCKSIQTLLYSSNQKNYLAW

QQKPGKA PKLLIYWASIRKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPLTFGGGTKLEIK >9E4Vκ Version2 (SEQ ID NO: 4)DIQMTQSPSSLSASVGDRVTITCKSIQTLLYSSNQKNYLAWYQQKPGKAPKLLIYWASIRKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPLTFGGGTKLEIK >9E4Vic Version3 (SEQ ID NO: 5)DIQMTQSPSSLSASVGDRVTITCKSIQTLLYSSNQKNYLAW

QQKPGKA PKLLIYWASIRKSGVPSRFSGSGSGTDFTLTISSLQPED

ATYYCQQYY SYPLTFGGGTKLEIK >9E4vh Version1 (SEQ ID NO: 8)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASISSGGGSTYYPDNVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCSRGGAGIDYWGQGTLVTVSS >9E4vh Version2 (SEQ ID NO: 9)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASISSGGGSTYYPDNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

R GGAGIDYWGQGTLVTVSS >9E4vh Version3 (SEQ ID NO: 10)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASISSGGGSTYYPDNVKGRFTISRD

AKNSLYLQMNSLRAEDTAVYYCAR GGAGIDYWGQGTLVTVSS

Kabat numbering for AAY33350 light chain and AAC50998 heavy chain arelisted below:

Kabat numbering for AAY33350 light chain:

L1 D L2 I L3 Q L4 M L5 T L6 Q L7 S L8 P L9 S L10 S L11 L L12 S L13 A L14S L15 V L16 G L17 D L18 R L19 V L20 T L21 I L22 T L23 C L24 R L25 A L26S L27 Q L28 S L29 I L30 S L31 S L32 Y L33 L L34 N L35 W L36 Y L37 Q L38Q L39 K L40 P L41 G L42 K L43 A L44 P L45 K L46 L L47 L L48 I L49 Y L50A L51 A L52 S L53 S L54 L L55 Q L56 S L57 G L58 V L59 P L60 S L61 R L62F L63 S L64 G L65 S L66 G L67 S L68 G L69 T L70 D L71 F L72 T L73 L L74T L75 I L76 S L77 S L78 L L79 Q L80 P L81 E L82 D L83 F L84 A L85 T L86Y L87 Y L88 C L89 Q L90 Q L91 S L92 Y L93 S L94 T L95 P L96 L L97 T L98F L99 G L100 G L101 G L102 T L103 K L104 L L105 E L106 I L107 K L108 —L109 — L110 — L111 —

Kabat numbering for AAC50998 heavy chain:

H1 E H2 V H3 Q H4 L H5 V H6 E H7 S H8 G H9 G H10 G H11 L H12 V H13 Q H14P H15 G H16 G H17 S H18 L H19 R H20 L H21 S H22 C H23 A H24 A H25 S H26G H27 F H28 T H29 F H30 S H31 S H32 Y H33 W H34 M H35 S H36 W H37 V H38R H39 Q H40 A H41 P H42 G H43 K H44 G H45 L H46 E H47 W H48 V H49 A H50N H51 I H52 K H52A Q H53 D H54 G H55 S H56 E H57 K H58 Y H59 Y H60 V H61D H62 S H63 V H64 K H65 G H66 R H67 F H68 T H69 I H70 S H71 R H72 D H73N H74 A H75 K H76 N H77 S H78 L H79 Y H80 L H81 Q H82 M H82A N H82B SH82C L H83 R H84 A H85 E H86 D H87 T H88 A H89 V H90 Y H91 Y H92 C H93 AH94 R H95 G H96 S H97 S H98 D H99 M H100 — H101 D H102 Y H103 W H104 GH105 Q H106 G H107 T H108 L H109 V H110 T H111 V H112 S H113 S H114 —

Kabat number of other heavy and light chain variable regions can bedetermined by alignment with corresponding residues assigned the samenumber or using commercially available software.

TABLE 4 Kabat numbering of preferred framework residues for backmutationin humanized 9E4 antibodies Humanized AAY33350 AAC50998 Mouse HumanizedHumanized Humanized 9E4 v4 light chain heavy chain 9E4 9E4 v1 9E4 v2 9E4v3 (heavy chain) L36 Y — F F Y F — L83 F — L F F L — H73 — N D D N D NH93 — A S S S A A

Example II. Passive Immunization with α-Synuclein Antibodies

The goal of this experiment is to determine effectiveness of α-synucleinantibodies in in vitro and in vivo studies as well as behavioral assays.We used α-synuclein transgenic (Line 61), α-synuclein knockout andwildtype female mice, 3-4 months old at initiation and n=14/group.Antibodies tested included 9E4 (IgG1, epitope: amino acids 118-126 ofalpha synuclein), 5C1 (IgG1, epitope: amino acids 118-126 of alphasynuclein, c-linker), 5D12, IgG2 (SN118-126), 1H7, IgG1 (SN 91-99) andan IgG1 control antibody 27-1. Mice received a dosage of 10 mg/kg over a5 month period, for a total of 21 injections. In addition, the animalswere injected with lentivirus (LV) expressing human α-synuclein (wt) byunilateral introduction of human α-synuclein (wt) into the hippocampus.

Readout antibodies include those from Chemicon (epitope: full-lengthalpha synuclein), Millipore (epitope: full-length alpha synuclein), andNeotope, ELADW 105 (epitope: amino acids 121-124 of full-length alphasynuclein).

Endpoints:

Antibody titers were measured during the in life phase. Behavioralassays include Morris Water Maze test (MWW) and horizontal beam test.The round beam test is a test of motor balance, coordination and gaitconducted using two beams of varying diameter. Beam A is the largerdiameter (easier, considered the training beam) and Beam D is thesmaller diameter (more difficult, considered the testing beam). Data ispresented as “errors” (number of slips/10 cm) and “speed” (time taken totravel 10 cm/sec). Water maze performance was carried out at weeks 10and termination. The following neuropathology measurements were taken:alpha synuclein aggregation, synaptophysin, and MAP2. The followingbiochemistry measurements were taken: alpha synuclein, PSD95,synaptophysin. Selected multilabeling and confocal labeling were carriedout using synaptic, neuronal and glial markers.

The results showed that all antibodies, except 5D12, producedsignificant reduction in α-syn accumulation and preservation of synapticand dendritic densities, as well as positive outcomes in MWMperformance. The 9E4 antibody is effective in in vitro and in vivostudies as well as behavioral assays. Readouts indicate antibody mayreduce neuritic/axonal alpha synuclein aggregates.

Behavioral Results:

The 9E4 antibody improved water maze performance in α-synucleintransgenic mice (FIGS. 3-4). In contrast, the 5D12 antibody did notimprove water maze performance in α-synuclein transgenic mice (FIG. 4).The 9E4 and 1H7 antibodies improved performance on the beam test asmeasured both by speed and errors, whereas the 5D12 and 5C1 antibodiesdid not (FIG. 4).

Neuropathology Results:

The 9E4, 1H7 and 5C1 antibodies reduced ELADW-105 positive neuriticdystrophy, whereas the 5D12 antibody did not. In alpha synucleintransgenic mice, the 9E4 antibody reduced the area of neuropil by 43% inneocortex and by 40% in basal ganglia as compared to control. The 9E4antibody also preserved synaptophysin and MAP2 in neocortex and basalganglia.

Example III. Immunoprecipitation

Immunoprecipitation was performed to test the binding efficacy ofvarious versions of humanized 9E4 antibodies towards its antigen takenfrom diseased tissue (FIG. 5). 150 μg of Tris-soluble brain lysates fromDementia with Lewy Body brains were immunoprecipitated with κ μg of eachindicated antibody using Protein G magnetic beads (New England Biolabs).Samples were washed 5 times with PBS/350 mM NaCl/0.5% NP-40, boiled, andthe resulting samples resolved by SDS-PAGE. After blotting, membraneswere incubated with Ab5038 (Millipore), a polyclonal antibody thatdetects total synuclein. The experiment was repeated three times toconfirm accuracy.

Example IV. Western Blot

Western blotting of recombinant human synuclein with mouse, chimeric andhumanized 9E4 antibodies is shown in FIG. 6. Antibody dilution curveswere apparently similar for mouse, chimeric and humanized 9E4antibodies. All antibodies detected a band at 28 KDa and a second bandat 49 kDa. The 49 kDa band is likely a multimer of synuclein.

Indicated amounts of recombinant, bacterially-expressed human wild-typesynuclein were resolved by SDS-PAGE, and blotted with identical amountsof the indicated form of 9E4. After washing, species-appropriate goatpolyclonal antibodies conjugated to the IRDye-800 fluorophor wereapplied, and the blot was washed. Exposure times were identical for thedifferent antibodies.

DEPOSIT

The following hybridoma has been deposited under the provisions of theBudapest Treaty with the American Type Culture Collection (ATCC, P.O.Box 1549, Manassas, Va. 20108) on the date indicated. This deposit willbe maintained at an authorized depository and replaced in the event ofmutation, nonviability or destruction for a period of at least fiveyears after the most recent request for release of a sample was receivedby the depository, for a period of at least thirty years after the dateof the deposit, or during the enforceable life of the related patent,whichever period is longest. All restrictions on the availability to thepublic of these cell lines will be irrevocably removed upon the issuanceof a patent from the application.

Monoclonal Date of Accession antibody Cell Line Epitope/SpecificityIsotype Deposit No. 9E4 JH17.9E4.3.37.1.14.2 alpha-synuclein IgG1κ Feb.26, PTA-8221 residues 118-126 2007

What is claimed is:
 1. A vector comprising a nucleic acid encoding amature heavy chain variable region and a mature light chain variableregion operably linked to one or more regulatory sequences to effectexpression of an antibody comprising the mature heavy chain variableregion and the mature light chain variable region that specificallybinds human alpha-synuclein, wherein the mature heavy chain variableregion comprises the three Kabat CDRs of SEQ.NO:11 and the mature lightchain variable region comprises the three Kabat CDRs of SEQ ID NO:4,wherein position L36 (Kabat numbering) is occupied by F, position L83(Kabat numbering) is occupied by L and position H73 (Kabat numbering) isoccupied by D.
 2. The vector of claim 1, wherein the mature heavy chainvariable region has the amino acid sequence of SEQ ID NO:10 and themature light chain variable region has the amino acid sequence of SEQ IDNO:5.
 3. The vector of claim 2, wherein the nucleic acid further encodesa heavy chain constant region fused to the mature heavy chain variableregion and a light chain constant region fused to the mature light chainvariable region.
 4. The vector of claim 3, wherein the heavy chainconstant region has the sequence of SEQ ID NO:32 with or without theC-terminal lysine and the light chain constant region has the sequenceof SEQ ID NO:13.
 5. The vector of claim 4, wherein the nucleic acid hasa sequence comprising SEQ ID NO:18 or 20 encoding the mature heavy chainvariable region.
 6. The vector of claim 4, wherein the nucleic acid hasa sequence comprising SEQ ID NO:17 encoding the mature light chainvariable region.
 7. The vector of claim 4, wherein the nucleic acid hasa sequence comprising SEQ ID NO:18 or 20 encoding the mature heavy chainvariable region and has a sequence comprising SEQ ID NO:17 encoding themature light chain variable region.
 8. The vector of claim 1, whereinthe antibody is a single chain antibody in which the heavy and lightchain mature variable regions are linked through a spacer.
 9. The vectorof claim 1, wherein the antibody is a Fab fragment.
 10. The vector ofclaim 1, wherein the one or more regulatory sequences include one ormore of a promoter, enhancer, ribosome binding site, and transcriptiontermination signal.
 11. The vector of claim 1, wherein the nucleic acidfurther encodes signal peptides fused to the mature heavy and lightchain variable regions.
 12. The vector of claim 1, wherein the nucleicacid is codon-optimized for expression in a host cell.
 13. The vector ofclaim 1, wherein the one or more regulatory sequences include aeukaryotic promoter.
 14. The vector of claim 1, wherein the nucleic acidfurther encodes a selectable gene.
 15. A host cell comprising a nucleicacid encoding a mature heavy chain variable region and a mature lightchain variable region operably linked to one or more regulatorysequences to effect expression of an antibody comprising the matureheavy chain variable region and the mature light chain variable regionthat specifically binds human alpha-synuclein, wherein the mature heavychain variable region comprises the three Kabat CDRs of SEQ.NO:11 andthe mature light chain variable region comprises the three Kabat CDRs ofSEQ ID NO:4, wherein position L36 (Kabat numbering) is occupied by F,position L83 (Kabat numbering) is occupied by L and position H73 (Kabatnumbering) is occupied by D.
 16. First and second vectors respectivelycomprising nucleic acids encoding a mature heavy chain variable regionand a mature light chain variable region, each operably linked to one ormore regulatory sequences to effect expression an antibody comprisingthe mature heavy chain variable region and the mature light chainvariable region, wherein the mature heavy chain variable regioncomprises the three Kabat CDRs of SEQ.NO:11 and the mature light chainvariable region comprises the three Kabat CDRs of SEQ ID NO:4, whereinposition L36 (Kabat numbering) is occupied by F, position L83 (Kabatnumbering) is occupied by L and position H73 (Kabat numbering) isoccupied by D.
 17. The vectors of claim 16, wherein the mature heavychain variable region has the amino acid sequence of SEQ ID NO:10 andthe mature light chain variable region has the amino acid sequence ofSEQ ID NO:5.
 18. The vectors of claim 17, wherein the nucleic acidsrespectively further encodes a heavy chain constant region fused to themature heavy chain variable region and a light chain constant regionfused to the mature light chain variable region.
 19. The vectors ofclaim 18, wherein the heavy chain constant region has the sequence ofSEQ ID NO:32 with or without the C-terminal lysine and the light chainconstant region has the sequence of SEQ ID NO:13.
 20. The vectors ofclaim 19, wherein the nucleic acids respectively have sequencescomprising SEQ ID NO:18 or 20 encoding the mature heavy chain variableregion and SEQ ID NO:17 encoding the mature light chain variable region.21. A host cell comprising nucleic acids encoding a mature heavy chainvariable region and a mature light chain variable region, each operablylinked to one or more regulatory sequences to effect expression anantibody comprising the mature heavy chain variable region and themature light chain variable region, wherein the mature heavy chainvariable region comprises the three Kabat CDRs of SEQ.NO:11 and themature light chain variable region comprises the three Kabat CDRs of SEQID NO:4, wherein position L36 (Kabat numbering) is occupied by F,position L83 (Kabat numbering) is occupied by L and position H73 (Kabatnumbering) is occupied by D.